T cell responses are governed by avidity and co-stimulatory thresholds

We analyzed the avidity and CD28-mediated co-stimulatory requirements for the activation of T cells in vivo and in vitro. The strength of the T cell/antigen-presenting cell interaction was varied by using altered peptide ligands for stimulation. Co-stimulatory requirements were studied using T cells from CD28-deficient mice. The results indicate that T cell activation is not an all-or-nothing event, but occurs in distinct steps. For each step, a certain avidity, co-stimulatory threshold or both, must be met. Depending upon the strength of the interaction between the T cell receptor and the major histocompatibility complex/peptide and the presence of CD28 co-stimulatory signals, T cells may undergo blast formation alone or proliferate or eventually both proliferate and differentiate to effector cells. Thus, T cell activation is governed by both avidity and co-stimulatory thresholds.     

Complex decay dynamics of HIV virions,intact and defective proviruses,and 2LTR circles following initiation of antiretroviral therapy

In persons living with HIV-1 (PLWH) who start antiretroviral therapy (ART), plasma virus decays in a biphasic fashion to below the detection limit. The first phase reflects the short half-life (<1 d) of cells that produce most of the plasma virus. The second phase represents the slower turnover (t_1/2 = 14 d) of another infected cell population, whose identity is unclear. Using the intact proviral DNA assay (IPDA) to distinguish intact and defective proviruses, we analyzed viral decay in 17 PLWH initiating ART. Circulating CD4⁺ T cells with intact proviruses include few of the rapidly decaying first-phase cells. Instead, this population initially decays more slowly (t_1/2 = 12.9 d) in a process that largely represents death or exit from the circulation rather than transition to latency. This more protracted decay potentially allows for immune selection. After ∼3 mo, the decay slope changes, and CD4⁺ T cells with intact proviruses decay with a half-life of 19 mo, which is still shorter than that of the latently infected cells that persist on long-term ART. Two-long-terminal repeat (2LTR) circles decay with fast and slow phases paralleling intact proviruses, a finding that precludes their use as a simple marker of ongoing viral replication. Proviruses with defects at the 5′ or 3′ end of the genome show equivalent monophasic decay at rates that vary among individuals. Understanding these complex early decay processes is important for correct use of reservoir assays and may provide insights into properties of surviving cells that can constitute the stable latent reservoir.

For persons living with HIV-1 (PLWH), lifelong adherence to antiretroviral therapy (ART) is critical for maintaining suppression of viral replication and forestalling the development of fatal immunodeficiency. Following initiation of ART, plasma virus levels decay rapidly to below the limit of detection of clinical assays (1–6). Because antiretroviral drugs block new infection of susceptible cells, but not virus production by cells that have an integrated viral genome, this decay must reflect the loss of productively infected cells, cells that were infected prior to the initiation of ART. Productively infected cells could die from viral cytopathic effects, cytolytic host effector mechanisms, or virus-independent T cell turnover. In principle, the decay of plasma virus could also be explained by transition to a nonproductive or latent state of infection. Importantly, the decay is biphasic, indicating the presence of two populations of productively infected cells with different half-lives. Most of the plasma virus is produced by cells that decay very rapidly, with a half-life of less than 1 d. Perelson et al. (4) showed that after most of these cells have decayed, the slope changes, reflecting the slower elimination of a second population of productively infected cells. This population decays with a variable half-life (mean ∼ 2 wk). Although this biphasic decay is a consistent feature of the response to ART, there is still uncertainty about the nature, anatomic location, and fate of the cells responsible for virus production during the first and second phases of decay (referred to here as first- and second-phase cells, respectively). The differences between these two populations have never been elucidated.The first and second phases of decay bring viremia down to below the limit of detection of clinical assays (typically 20 to 50 copies of HIV-1 RNA per mL of plasma) within months of ART initiation, initially raising hope for eradication. However, a latent form of the virus persists in resting memory CD4⁺ T cells (7–14). Initial studies used a quantitative viral outgrowth assay (QVOA) to demonstrate that latently infected resting CD4⁺ T cells purified from PLWH on long-term suppressive ART could be induced to produce replication-competent virus by global T cell activation (8, 9). Longitudinal studies using the QVOA demonstrated that the half-life of the latent reservoir in resting CD4⁺ T cells is 44 mo in PLWH who are adherent to ART. This half-life is long enough to guarantee lifetime persistence of HIV-1 despite ART (12–14). Strategies targeting the latent reservoir in resting CD4⁺ T cells are a major focus of HIV cure research (15–17). In addition to resting CD4⁺ T cells, other cell types may contribute to HIV-1 persistence (18–20).Prior to and immediately following initiation of ART, the frequency of latently infected cells detected by QVOA is substantially higher than frequencies observed in PLWH on long-term ART (21). In principle, several different types of decay processes occurring over the first 6 to 12 mo of treatment could reduce the frequency of latently infected cells to the more stable frequencies observed in PLWH on long-term ART. Early studies by Jerome Zack and Mario Stevenson demonstrated that infected resting CD4⁺ T cells could harbor linear, unintegrated HIV-1 DNA in a state of preintegration latency (22, 23). Following cellular activation, linear unintegrated HIV-1 DNA can be integrated and transcribed, allowing production of virus (22, 23). The half-life of linear, unintegrated forms of the viral genome is not clear, with some studies suggesting that these forms are labile (22, 24–26). Some reverse-transcribed viral genomes can undergo homology-dependent or end-to-end ligation, generating one-long-terminal repeat or two-long-terminal repeat (2LTR) circles, respectively (reviewed in ref. 27). The stability of these forms is also controversial, but they are clearly replication-defective (27–31). Following integration of linear viral cDNA, decay dynamics depend on dynamics of the infected host cells, which can be eliminated by viral cytopathic effects, immune cytolytic effector mechanisms, and normal contraction-phase death of previously activated CD4⁺ T cells (32, 33).While the QVOA provides a definitive minimal estimate of the frequency of latently infected cells, it underestimates reservoir size because not all proviruses in resting CD4⁺ T cells are induced upon one round of maximum T cell activation (34–36). Many replication-competent proviruses require multiple rounds of stimulation for induction. As an alternative to the QVOA, many studies use PCR-based assays to measure proviral DNA. However, the vast majority of HIV-1 proviruses are defective due to apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide-like (APOBEC)-mediated hypermutation or large internal deletions (34, 37–39). PCR-based assays do not distinguish between defective and intact proviruses (40, 41). Although infected cell dynamics have been explored using PCR-based assays (42), the results likely reflect the dynamics of defective proviruses (41). The recently developed intact proviral DNA assay (IPDA) uses two carefully chosen amplicons to probe informative regions of individual proviruses to provide better discrimination between intact and defective proviruses (41, 43). This assay has proven useful in evaluating the long-term dynamics of cells with intact and defective proviruses, demonstrating differences in decay rates that may reflect some vulnerability of cells with intact proviruses to immune effector mechanisms (41, 44, 45).In this study, we use the IPDA to explore the decay of intact and defective proviruses at early time points following initiation of ART. We identify decay processes occurring over intermediate time scales, but with pronounced differences between intact and defective proviruses. Of particular importance is the second-phase decay because infected cells that survive second-phase decay may down-regulate HIV-1 gene expression and enter the stable latent reservoir. Our findings also provide insight into mechanisms for the elimination of the cells with intact viral genomes and into the proper use of assays for the latent reservoir.     

Free Zinc as a Predictive Marker for COVID-19 Mortality Risk

Free zinc is considered to be the exchangeable and biological active form of zinc in serum, and is discussed to be a suitable biomarker for alterations in body zinc homeostasis and related diseases. Given that coronavirus disease 2019 (COVID-19) is characterized by a marked decrease in total serum zinc, and clinical data indicate that zinc status impacts the susceptibility and severity of the infection, we hypothesized that free zinc in serum might be altered in response to SARS-CoV-2 infection and may reflect disease severity. To test this hypothesis, free zinc concentrations in serum samples of survivors and nonsurvivors of COVID-19 were analyzed by fluorometric microassay. Similar to the reported total serum zinc deficit measured by total reflection X-ray fluorescence, free serum zinc in COVID-19 patients was considerably lower than that in control subjects, and surviving patients displayed significantly higher levels of free zinc than those of nonsurvivors (mean ± SD; 0.4 ± 0.2 nM vs. 0.2 ± 0.1 nM; p = 0.0004). In contrast to recovering total zinc concentrations (r = 0.706, p < 0.001) or the declining copper–zinc ratio (r = −0.646; p < 0.001), free zinc concentrations remained unaltered with time in COVID-19 nonsurvivors. Free serum zinc concentrations were particularly low in male as compared to female patients (mean ± SD; 0.4 ± 0.2 nM vs. 0.2 ± 0.1 nM; p = 0.0003). This is of particular interest, as the male sex is described as a risk factor for severe COVID-19. Overall, results indicate that depressed free serum zinc levels are associated with increased risk of death in COVID-19, suggesting that free zinc may serve as a novel prognostic marker for the severity and course of COVID-19.     

Inhibition of TCR triggering by a spectrum of altered peptide ligands suggests the mechanism for TCR antagonism

Understanding the parameters involved in T cell activation has been complicated by the discovery of partial T cell agonists. Altered peptide ligands (APL) have recently shown that different subsets of T cell responses can be selectively activated by certain peptides, which define a hierarchy of T cell activation. For cytotoxic T cells, this hierarchy ranges from sensitizing target cells for lysis through proliferation to effector cell induction. The degree of TCR down-regulation mediated by APL-MHC interactions correlates well with the induction of specific T cell effector functions. This suggests that the potential agonist response induced by a given peptide occurs at different triggering thresholds. To examine the relative agonist and antagonist functions of different peptides, we have investigated the ability of lymphocytic choriomeningitis virus glycoprotein-derived APL to induce or inhibit a range of effector functions in naive CD8⁺ T cells. By this, we have defined a hierarchy of peptides that display a range of properties from strong agonist to no agonist function. At each level, peptides that were ranked lower in this hierarchy were able to interfere or antagonize the induction of effector functions by higher ranking peptides. We have therefore shown that this spectrum of peptides ranging from strong to no agonist function has an inverse gradient from strong antagonist to no antagonist function. Moreover, the ability of the different peptides to inhibit TCR internalization correlated with their ranking within the hierarchy. These findings support the model that antagonists are effectively preventing TCR oligomerization and functional TCR triggering.     

Brain biopsy in the diagnosis of cerebral mycosis fungoides

A case of cerebral mycosis fungoides co-existing with progressive multifocal leucoencephalopathy presented with dementia. Brain biopsy established the diagnosis of mycosis fungoides after cerebrospinal fluid examinations and computerised tomographic scanning of the brain produced non-specific abnormalities.     

Polyclonal rearrangements of the T-cell receptor β-chain in fatal angioimmunoblastic lymphadenopathy

Genomic rearrangement of germline T-cell antigen receptor (TcR) and immunoglobulin (Ig) genes was studied by Southern blot analysis in seven patients with angioimmunoblastic lymphadenopathy (AILD). In three cases clinically suspected of transformation into malignant lymphoma, hybridization with the TcRβ probe showed markedly dimished intensity in the 11.5 kb germline band after Eco RI digestion and normal germline configuration after Hind III and Bam HI digestion, indicating polyclonal T cell rearrangements. A clonal rearrangement of the TcRβ gene was detected in only one case at initial biopsy. No monoclonal rearrangement of Ig genes was observed. These data show that in some cases of AILD disease progression is indicated by polyclonal TcR rearrangements and not by outgrowth of a malignant clone, supporting the concept of AILD as an immunoregulatory disorder.     

Dissection of seroreactivity against the tryptophan-rich motif of the feline immunodeficiency virus transmembrane glycoprotein

Immunogenicity of the tryptophan-rich motif (TrpM) in the membrane-proximal ectodomain of the transmembrane (TM) glycoprotein of feline immunodeficiency virus (FIV) was investigated. Peptide 59, a peptide containing the TrpM of the TM of FIV, was covalently coupled to Qbeta phage virus-like particles (Qbeta-59) in the attempt to induce potent anti-TrpM B cell responses in cats. All Qbeta-59 immunized cats, but not cats that received a mixture of uncoupled Qbeta and peptide 59, developed antibodies that reacted with a same epitope in extensive binding and binding competition assays. The epitope recognized was composed of three amino acids, two of which are adjacent. However, Qbeta-59-immune sera failed to recognize whole FIV in all binding and neutralization assays performed. Furthermore, no reactivity against the TrpM was detected by screening sera from FIV-infected cats that had reacted with TM peptides, confirming that this epitope does not seem to be serologically functional in the FIV virion. The data suggest that TrpM may not be a suitable target for antiviral vaccine design.